NMR solution structure of the antifungal protein from Aspergillus giganteus: evidence for cysteine pairing isomerism

• Published in: Biochemistry (Easton) 1995-03, Vol.34 (9), p.3009-3021
• Main Authors: Campos-Olivas, Ramon, Bruix, Marta, Santoro, Jorge, Lacadena, Javier, Martinez del Pozo, Alvaro, Gavilanes, Jose G, Rico, Manuel
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; ASPERGILLUS; Aspergillus - chemistry; Aspergillus - genetics; Aspergillus giganteus; Binding Sites; CISTEINA; CYSTEINE; Cysteine - chemistry; ENERGIA; ENERGIE; ESPECTROMETRIA; Fungal Proteins - chemistry; Fungal Proteins - genetics; Isomerism; Magnetic Resonance Spectroscopy; MATEMATICAS; MATHEMATIQUE; Models, Molecular; Molecular Sequence Data; Molecular Structure; PESO MOLECULAR; POIDS MOLECULAIRE; PROPIEDADES ANTIMICOSICAS; PROPRIETE ANTIFONGIQUE; Protein Conformation; Protein Structure, Secondary; PROTEINAS; PROTEINE; Protons; SOLUCION; SOLUTION; Solutions; SPECTROMETRIE; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00009a032

The solution structure of the antifungal protein (AFP, 51 residues, 4 disulfide bridges) from Aspergillus giganteus has been determined by using experimentally derived interproton distance constraints from nuclear magnetic resonance (NMR) spectroscopy. Complete sequence-specific proton assignments were obtained at pH 5.0 and 35 degrees C. A set of 834 upper limit distance constraints from nuclear Overhauser effect measurements was used as input for the calculation of structures with the program DIANA. An initial family of 40 structures calculated with no disulfide constraints was used to obtain information about the disulfide connectivities, which could not be determined by standard biochemical methods. Three possible disulfide patterns were selected and the corresponding disulfide constraints applied to generate a family of 20 DIANA conformers for each pattern. Following energy minimization, the average pairwise RMSD of the 20 conformers of each family is 1.01, 0.89, and 1.01 angstroms for backbone atoms and 1.82, 1.74, and 1.81 angstroms for all heavy atoms. One of these three families contains the disulfide bridge arrangement actually present in the solution structure of AFP. Although the three families fulfill the NMR constraints, one of the disulfide patterns considered (cysteine pairs 7-33, 14-40, 26-49, 28-51) is favored among the others on the basis of previous chemical studies. It thus probably corresponds to the actual pattern of disulfide bridges present in the protein, and the corresponding family represents the solution structure of AFP. The folding of AFP consists of five antiparallel beta strands connected in a -1, -1, +3, +1 topology and highly twisted, defining a small and compact beta barrel stabilized by four internal disulfide bridges. A cationic site formed by up to three lysine side chains adjacent to a hydrophobic stretch, both at the protein surface, may constitute a potential binding site for phospholipids